Zoology 140 (2020) 125770

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Zoology

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Sperm length variation among Afrotropical songbirds reﬂects phylogeny rather than adaptations to the tropical environment T

Taiwo C. Omotorioguna,b,c, Tomáš Albrechtd,e, Jostein Gohlia, David Hořáke, Lars Erik Johannessena, Arild Johnsena, Jakub Kreisingere, Petter Z. Markia,f, Ulf Ottossonb, Melissah Rowea,g,h, Ondřej Sedláčeke, Jan T. Lifjelda,* a Natural History Museum, University of Oslo, P.O. Box 1172 Blindern, NO-0318 Oslo, Norway b A.P. Leventis Ornithological Research Institute, University of Jos, P.O. Box 13404, Nigeria c Biotechnology Unit, Department of Biological Sciences, Elizade University, P.M.B. 002, Ilara-Mokin, Nigeria d Institute of Vertebrate Biology, Academy of Sciences of the Czech Republic, v.v.i., Květná 8, Brno, CZ-60305, Czech Republic e Faculty of Science, Charles University, Prague, Viničná 7, CZ-12844, Czech Republic f Center for Macroecology, Evolution and Climate, Natural History Museum of Denmark, University of Copenhagen, Universitetsparken 15, DK-2100 Copenhagen, Denmark g Centre for Ecological and Evolutionary Synthesis, Department of Biosciences, University of Oslo, P.O. Box 1066 Blindern, NO-0316 Oslo, Norway h Department of Animal Ecology, Netherlands Institute of Ecology (NIOO-KNAW), 6700 AB, Wageningen, the Netherlands

ARTICLE INFO ABSTRACT

Keywords: Sperm cells vary tremendously in size and shape across the animal kingdom. In songbirds (Aves: Passeri), sperm Sperm have a characteristic helical form but vary considerably in size. Most of our knowledge about sperm morphology size in this group stems from studies of species in the Northern temperate zone, while little is known about the Passerides numerous species in the tropics. Here we examined sperm size in 125 Afrotropical songbird species with em- Phylogenetic effect phasis on the length of the major structural components (head, midpiece, flagellum), and total sperm length Speciation measured using light microscopy. Mean total sperm length varied from 51 μm to 212 μm across species. Those Comparative analyses belonging to the Corvoidea superfamily had relatively short sperm with a small midpiece, while those of the three major Passeridan superfamilies Passeroidea, Muscicapoidea and Sylvioidea showed large interspecific variation in total sperm length and associated variation in midpiece length. These patterns are consistent with previous findings for temperate species in the same major clades. A comparative analysis with songbird species from the Northern temperate zone (N = 139) showed large overlap in sperm length ranges although certain temperate families (e.g. Parulidae, Emberizidae) typically have long sperm and certain Afrotropical families (e.g. Cisticolidae, Estrildidae) have relatively short sperm. Afrotropical and temperate species belonging to the same families showed no consistent contrasts in sperm length. Sperm length variation among Afrotropical and Northern temperate songbirds exhibits a strong phylogenetic signal with little or no evidence for any directional latitudinal effect among closely related taxa.

1. Introduction among males for fertilizations (Parker, 1970), must play an important role (Pitnick et al., 2009; Simmons and Fitzpatrick, 2012). Changes in Sperm show extraordinary morphological diversity across the an- sperm length are also associated with changes in the architecture of the imal kingdom (Pitnick et al., 2009). For example, sperm length varies in female reproductive tract (Briskie et al., 1997; Miller and Pitnick, 2002; insects from ∼7 μm in a parasitoid wasp (Uzbekov et al., 2017)to Higginson et al., 2012). The theory of anisogamy generally emphasizes ∼58,290 μm in a fruit ﬂy(Pitnick et al., 1995). The evolutionary forces the selective advantage for males to produce tiny gametes in vast underlying this diversiﬁcation are not well understood although it is numbers (e.g., Parker, 1982; Lessells et al., 2009). Overall, there is good widely acknowledged that sperm competition, i.e. the competition evidence for sperm competition across the animal kingdom and that

⁎ Corresponding author. E-mail addresses: [email protected] (T.C. Omotoriogun), [email protected] (T. Albrecht), [email protected] (J. Gohli), [email protected] (D. Hořák), [email protected] (L.E. Johannessen), [email protected] (A. Johnsen), [email protected] (J. Kreisinger), [email protected] (P.Z. Marki), [email protected] (U. Ottosson), [email protected] (M. Rowe), [email protected] (O. Sedláček), [email protected] (J.T. Lifjeld). https://doi.org/10.1016/j.zool.2020.125770 Received 16 November 2019; Received in revised form 7 March 2020; Accepted 9 March 2020 Available online 23 March 2020 0944-2006/ © 2020 The Author(s). Published by Elsevier GmbH. This is an open access article under the CC BY license (http://creativecommons.org/licenses/BY/4.0/). T.C. Omotoriogun, et al. Zoology 140 (2020) 125770 males enhance their success in sperm competition through an increase after a gentle massage (Wolfson, 1952; Kleven et al., 2008; Kucera and in sperm numbers, i.e. the raffle principle (Parker, 1982, Pizzari and Heidinger, 2018) and diluted in a small volume (∼20 μl) of Phosphate- Parker, 2009). However, it does not automatically follow that sperm buffered saline (PBS) solution before fixed in ∼300 μl of 5% for- competition selects for smaller sperm. Empirical evidence suggests that maldehyde solution for later slide preparation. Sampled birds were the effect of sperm competition on sperm size evolution can be rather fitted with uniquely numbered aluminium bands (from SAFRING) to complex (Snook, 2005) and comparative analyses of many animal taxa avoid resampling, and immediately released into site of capture after have in fact revealed a positive association between sperm competition sampling. and sperm length (Fitzpatrick and Lüpold, 2014). There is also experimental evidence in insects that sperm competition selects for an increase in sperm length (Godwin et al., 2017). In passerine birds, 2.2. Species coverage sperm competition accelerates the rate of sperm length evolution, but changes in sperm length can go either way (Rowe et al., 2015). This Altogether, we analysed sperm data from 2937 males of 125 species means that sperm competition may select for both sperm number and from 28 taxonomic families from Africa. They all belong to four su- larger or smaller sperm, and that the trade-off between numbers and perfamilies of the Passeri clade (songbirds): Corvoidea (8 species from 4 size may vary considerably among taxa (Immler et al., 2011). families), Passeroidea (52 species from 7 families), Muscicapoidea (15 We report on a study of sperm length variation among Afrotropical species from 2 families), and Sylvioidea (50 species from 15 families). songbirds (Aves: Passeriformes: Passeri). Across all songbird species, The Corvoidea belongs to the infraorder Corvides, whereas the three regardless of geographic location, sperm length varies in the range of 40 other superfamilies belong to the Passerides (sensu Cracraft, 2014). For – 290 μm, which is a modest size range compared to insects, but still the comparison with species of the Northern temperate zone, we much wider than in other groups of birds (Jamieson, 2007). Most compiled a data set from the Avian Sperm Collection Database at the knowledge of variation in sperm traits across the global avifauna ori- Natural History Museum, University of Oslo (NHMO) (Lifjeld, 2019). ginates from studies of birds in the Northern temperate zone with little This data set contained 139 species from 34 families, all belonging to known from the tropics. This might imply a biased picture as ecological the same two infraorders Corvides and Passerides as the Afrotropical requirements and adaptation may affect trait evolution differently in species. The two data sets, with sperm length measurements averaged the two regions, especially the evolution of various reproductive traits for species and the raw data from individual specimens, are available as and mating systems (Stutchbury and Morton, 2008), and hence the supplementary material (Additional file 2). The data set of sperm evolution of sperm. Life-history traits show some differences between measurements with metadata can also be downloaded from GBIF.org tropical and temperate birds in adult mortality (Peach et al., 2001), (Omotoriogun et al., 2020). reproductive rates and clutch size (Ricklefs and Wikelski, 2002), pace of life (Wiersma et al., 2007) and parental investment (Ghalambor and Martin, 2001; Jetz et al., 2008; McNamara et al., 2008). There is also 2.3. Species phylogeny correlative evidence showing associations between various life-history and socioecological traits and proxies for sperm competition in birds The phylogenetic trees of songbird species used for analyses were (reviewed in Lifjeld et al., 2019), from which one might predict that pruned from a larger tree (Marki, 2018) which was obtained using a tropical species in general should have lower levels of sperm competi- supermatrix approach (Sanderson et al., 1998). DNA sequences of tion than those in the temperate zones (Stutchbury and Morton, 2001). species were downloaded from GenBank; where sequence data was However, the available evidence for a contrast in sperm competition is unavailable from focal species a substitute sequences from close re- far from conclusive (Macedo et al., 2008; Albrecht et al., 2013; Lifjeld latives was used instead (sensu Price et al., 2014). The sequences ori- et al., 2019). ginate from nuclear intron 2 of the myoglobin gene (Myo2), introns 6 There are some comparative studies on sperm lengths in African and 7 of the ornithine decarboxylase (ODC) gene, intron 11 of the birds (Omotoriogun et al., 2016a, b) including those that list sperm glyceraldehyde-3-phosphodehydrogenase (GAPDH) gene, and the two length data for African species (Immler et al., 2011, Albrecht et al., mitochondrial genes cytochrome b (cytb) and NADH dehydrogenase 2013), but a more comprehensive analysis of variation in sperm length subunit 2 (ND2). A matrix of the GenBank accession numbers is avail- in Afrotropical birds relative to Northern temperate birds is lacking. able as supplementary material (Additional file 4). Here we present the first comparative analysis of this kind based on 125 Sequences alignment was performed using the Muscle algorithm Afrotropical species, and combined this with data on 139 temperate (Edgar, 2004) in SeaView v4.5.4 (Gouy et al., 2009); ambiguous regions species of songbird to investigate whether they follow the general contained in the resulting alignments were removed using GBlocks patterns known for temperate species of the same clade or whether (Castresana, 2000). The data matrix of all five genes (3240 bp) was there are specific patterns to Afrotropical species. concatenated and analysed in RAxML v8.2.4. The best fitting model of nucleotide evolution was determined by the Bayesian Information 2. Materials and methods Criterion (BIC) in jModelTest 2 (Darriba et al., 2012) and applied as follows: GTR + I + Γ model for cytb, ND2, and ODC; GTR + Γ for 2.1. Field work Myo2, and SYM + I + Γ for GAPDH partitions. We applied relaxed uncorrelated log-normal distribution for the molecular clock models Our study samples were collected from birds in Nigeria and that was unlinked across all five gene partitions (Drummond et al., Cameroon, specifically at Amurum Forest Reserve, Jos (09° 53′ N, 08° 2006). A Yule speciation process was used for the tree prior, with rate 59′ E); Yankari Game Reserve, Bauchi (09° 50′ N, 10° 30′ E); Omo Forest heterogeneity, base frequencies, and substitution rates were unlinked Reserve, Ogun (06° 51′ N, 4° 30′ E), Okomu National Park, Benin (06° across the five gene partitions. In BEAST, Markov Chain Monte Carlo 15′ N, 05° 09′ E); and IITA Forest, Ibadan (07° 30′ N, 03° 55′ E); Mt chains was ran for 100 million generations, sampling every 10 000 Cameroon (primary lowland to montane forest; 04° 15′ N, 09° 09′ E), generations. Convergence diagnostics based on Effective Sample Size Big Babanki, and Laide Farm Bamenda-Banso Highlands (forest-farm- was assessed in Tracer v1.6 (Rambaut et al., 2014) discarding 10% as land mosaics; 06° 05′ N, 10° 28′ E). Birds were captured using mist-nets burn-in. Results were summarized as posterior distribution as a max- (assisted with song-playback), and breeding males were sampled i.e., imum clade credibility tree (with mean node heights) using TreeAn- February to October in 2010–2015 in Nigeria, and November to June in notator v1.8.3 (Drummond et al., 2012). A nexus file for this tree, 2008–2014 in Cameroon. Sperm samples (∼ 0.5-3 μl) were collected containing all 264 species included in this study, is available as a with microcapillary tubes from the cloacal protuberance of male birds supplementary material (Additional file 1).

2 T.C. Omotoriogun, et al. Zoology 140 (2020) 125770

Fig. 1. The sperm component measurements. The length of the three components, head, midpiece and tail, were measured separately, the length of the ﬂagellum was calculated as the sum of midpiece and tail, and total sperm length was calculated as the sum of head and ﬂagellum. The sperm image is from a Blue- billed Malimbe Malimbe nitens.

2.4. Measurement of sperm morphology λ = 0. We also restricted analysis to family levels with species representation from both regions, and tested the effect of region on total We prepared microscope glass slides from a small aliquot (∼15 μl) sperm length, and length of sperm components. PGLS regressions were of the formaldehyde fixed sperm samples by spreading a drop of the performed using the package ”caper” (Orme, 2013). fixed sperm sample on a clean microscope slide and let it air-dry for 24 hours. Slides were then rinsed with distilled water and allowed to air-dry. Using a digital camera (DFC420, Leica Microsystems, 3. Results and Discussion Heerbrugg, Switzerland) mounted onto a digital light microscope μ (DM6000 B, Leica Microsystems), high-resolution digital images of in- For Afrotropical songbirds, sperm lengths ranged from 51 m in the μ dividual spermatozoon were captured at microscope magnifications Singing Cisticola Cisticola cantans to 212 m in the Streaky-headed (160× or 320×). Leica application suite version 2.6.0 R1 was applied Seedeater Crithagra gularis. Among the temperate species, sperm lengths μ μ to measure ( ± 0.1 μm) the length of sperm head, midpiece and tail (i.e. ranged from 43 m (White-throated Dipper Cinclus cinclus) to 280 m the section of the flagellum not entwined by the midpiece) following (Indigo Bunting Passerina cyanea, Eastern Towhee Pipilo ery- Laskemoen et al. (2007). We calculated sperm total length (sum of throphthalmus and Chipping Sparrow Spizella passerina). A histogram head, and midpiece, and tail length) and flagellum length (sum of covering all species of the two regions illustrates the more restricted midpiece and tail length). The measured sperm components are illu- range of sperm lengths for Afrotropical species, and that sperm on strated in Fig. 1. Repeated measurements of the same 15 sperm cells average across all species are shorter for Afrotropical than for Northern from a single individual showed high repeatability (head: r = 0.87, temperate species (Fig. 2). When the Afrotropical species were broken down by the four su- F14,15 = 14.75, P < 0.001; midpiece: r = 0.81, F14,15 = 9.76, P < 0.001, tail: r = 0.83, F = 10.94, P < 0.001), as calculated perfamilies (see example images in Fig. 3), sperm were relatively short 14,15 – μ according to Lessells and Boag (1987). Measurements of sperm head, in Corvoidea (54 83 m) compared to the more diverse sperm lengths – μ midpiece, flagellum and total length for individual males were based on in the three Passerides superfamilies, i.e. Passeroidea (53 212 m), – μ – μ the mean of 10 spermatozoa measured per male as a standard (mean of Muscicapoidea (81 184 m) and Sylvioidea (51 148 m). These 9.8, minimum of 3 sperm measured in samples with few sperm). patterns of sperm length variation among African songbirds largely match the patterns seen among the major clades, i.e. above the family level, in the Passeri (Jamieson, 2007); longer sperm (> 100 μm) are 2.5. Statistics and comparative analysis typically found within the Passerides songbirds, while Corvides songbirds have shorter sperm, similar to the sister group of the Passeri, the Analyses were performed in R version 3.1.2 (R Development Core Tyranni (Jamieson, 2007). It suggests that longer sperm have evolved Team, 2017). Prior to analysis, we log-transformed data for all sperm frequently within each of the superfamilies of the Passerides, from an traits. We reconstructed ancestral character states of sperm total length using the “phytools” (Revell, 2013) and the “ape” package (Paradis and Schliep, 2018) in R. Character states were estimated at internal nodes using maximum likelihood with “fastAnc” and “contMap” functions, both in” phytools” (Revell, 2013). We tested for the presence of phylogenetic signal in sperm traits using Pagel’s λ (Pagel, 1999) and Blomberg’s K (Blomberg et al., 2003) in “phytools” (Revell, 2013). These measures of phylogenetic signal are not identical, λ measures the strength of phenotypic-genotypic covar- iance assuming Brownian motion (λ = 1 equals Brownian motion) whereas K reflects the partitioning of trait variance among and within clades: with high K values implying more variance among clades and low K values meaning more variance among the terminal branches. For Pagel’s λ, log-likelihood ratio tests were used to determine if the estimated maximum likelihood values of λ differed from 0 (no phylogenetic signal) or 1 (total dependence on phylogeny); whereas randomi- zation test were used to determine whether traits exhibited a significant phylogenetic signal (K > 0) in Blomberg’s K. We performed phylogenetic generalized least squares (PGLS) regressions to test for differences in sperm traits (as response variable) between Afrotropical and Northern temperate (region as predictor variable). The PGLS approach accounts for the statistical non-in- dependence of data points as a result of common ancestry of species Fig. 2. Histogram of total sperm lengths of songbird species from the (Pagel, 1999; Freckleton et al., 2002) and allows the estimation (via Afrotropical and Northern temperate zones, the dash lines representing the maximum likelihood) of the phylogenetic scaling parameter lambda (λ) mean of sperm length (Afrotropical, mean =93.6 μm, N = 125; Northern μ as above. We tested the likelihood ratio of λ value against λ = 1 and temperate, mean = 134.0 m, N = 139).

3 T.C. Omotoriogun, et al. Zoology 140 (2020) 125770

Fig. 3. Examples of sperm length variation across four superfamilies of Afrotropical songbirds illustrated by digital images scaled to the same magnification (the 40 μm scalebar indicated). The 27 species are: Muscicapoidea: M1-Turdus pelios, M2-Cossypha albicapillus, M3-Cossypha niveicapilla, M4-Neocossyphus poensis, M5- Melaenornis pallidus, M6-Stiphrornis erythrothorax. Passeroidea: P1-Vidua macroura, P2-Emberiza affinis, P3-Emberiza tahapisi, P4-Ploceus cucullatus, P5-Ploceus heuglini, P6-Euplectes macroura, P7-Cryptospiza reichenovii, P8- Cinnyris venustus, P9- Cinnyris cupreus. Sylvioidea: S1-Cisticola guinea, S2-Cercotrichas harlaubi, S3- Sylvietta brachyura, S4- Eremomela pusilla, S5-Prinia subflava, S6-Psalidoprocne obscura, S7-Phyllastrephus albigularis, S8-Pycnonotus barbatus, S9-Nicator chloris. Corvoidea: C1- Platysteira cyanea, C2-Terpsiphone rufiventer, C3-Dyaphorophyia blissetti. ancestral state of short sperm. family stand out as having a longer sperm head than the rest. Figures When the sperm lengths are mapped onto the phylogeny (Fig. 4), it visualizing the length of the different sperm components mapped onto becomes evident that certain clades stand out with some distinct pat- the phylogeny (as in Fig. 4) can be found in the supplementary material terns in sperm lengths. For example, the Fringillidae and Muscicapidae (Additional file 3). have longer sperm whereas the Cisticolidae have consistently shorter There was no effect of region (Afrotropical versus Northern tem- sperm than all the other families. Within the Ploceidae, species of the perate zone) on overall sperm length or the length of sperm components genus Euplectes have consistently longer sperm than those of their sister when controlling for phylogeny (Table 1). The skewed distributions genus Ploceus (Fig. 4). There are, however, also some large intrageneric apparent in Fig. 2 must therefore be largely due to family level differ- contrasts in sperm lengths among Afrotropical species, such as Crithagra ences in sperm length and uneven family representation from the two gularis (Fringillidae) and Lagonosticta sanguinodorsalis (Estrildidae) regions. Some African families (e.g. Cisticolidae, Estrildidae) typically having rather long sperm compared to their congenerics. have short sperm while some Northern temperate families (e.g. Em- Another major contrast between Corvides and Passerides sperm is berizidae, Parulidae) have rather long sperm. There was also a strong the length of the midpiece. It is short in Corvides and long in Passerides, phylogenetic signal in sperm length components across Afrotropical except for the atypical Passerides sperm in some Pyrrhula finches and Northern temperate birds as revealed by Pagel’s λ (λ ≥ 0.884, (Lifjeld et al., 2013). The long midpiece is strongly correlated with the P < 0.0001) and Blomberg’sK(K ≥ 0.893, P = 0.001) (Table 1). flagellum length in Passerides, and covers up to almost 90% of total Midpiece, flagellum and total sperm length tended to be more similar sperm length in the species with the longest sperm (Lifjeld, 2019). The among related species than expected under Brownian motion but with length of the sperm head is not much differentiated across the Passer- certain marked exceptions (e.g. Crithagra gularis and Lagonosticta san- iformes. In the present data set, only members of the Muscicapidae guinodorsalis). Most of the variation in sperm length can therefore be

4 T.C. Omotoriogun, et al. Zoology 140 (2020) 125770

Fig. 4. Variations in total sperm length (μm) in Afrotropical (N = 125) and Northern temperate (N = 139) songbird mapped on their phylogeny. Sperm length is visualized in colour along the branches and nodes in the phylogeny in a gradient from red (short sperm) to blue (longer sperm). Branch lengths are given in million years (myr). The barplots on the per- iphery of the phylogeny show sperm lengths for species of the two regions separated by colour of the bars (Afrotropical = red, Northern temperate = black).

attributed to differences among the deeper nodes in the phylogeny, e.g. 4. Authors’ contribution the family level. When we restricted the analysis to four larger families (Fringillidae, Conceived and designed the study: TCO, JTL; data collection: all Hirundinidae, Muscicapidae, and Turdidae) with multiple species re- authors; data analyses: TCO; data management and interpretation: presented in both regions, there was still no significant effect of region TCO, JTL, LEJ, drafted the manuscript: TCO; All authors read, com- on sperm length or sperm components (Fig. 5, Table 2). The only ex- mented and approved the final manuscript. ception was head length in the Turdidae for the separate family analysis, but this could be an artefact of multiple tests. Again, there were strong phylogenetic signals in total sperm length and the length of the 5. Availability of data and materials different sperm components (Table 2). We therefore conclude that the large variation in sperm length ob- The datasets for this article are included within the article and its served among songbirds has a strong phylogenetic component and that additional supporting files. The dataset of sperm measurements is also much of the differentiation occurred early in the evolutionary history of published on GBIF.org (Omotoriogun et al., 2020). Sperm samples are the clade. There are marked differences in sperm length between deposited in the Avian Sperm Collection at Natural History Museum, taxonomic families, which explains the tendency for Afrotropical spe- Oslo (Lifjeld, 2019), which can be accessed online at the museum’s cies to have shorter sperm than Northern temperate species. We find no website (https://www.nhm.uio.no/). evidence that current selection pressures related to the tropical environment and associated adaptations in ecology and life history, can explain any differences in sperm length among closely related species. 6. Consent for publication Nonetheless, we must emphasize that species coverage was not ex- haustive for this study, as sperm morphology remains unknown for the Not applicable majority of Afrotropical passerines.

Table 1 Phylogenetic least squares (PGLS) analysis of diﬀerences in sperm components between Afrotropical (N=125) and temperate (N=139) species, with the component of interest as a response variable and climate zone as explanatory variable. The model including the maximum-likelihood of lambda (λ) value was compared against the models assuming λ = 1 and λ = 0, and superscripts following the λ values indicate probability (P) of likelihood-ratio of sperm trait (ﬁrst position: against λ =0; second position: against λ = 1). The table also gives two estimates of the phylogenetic signal in the sperm components as expressed by Pagel’s λ (with P based on a likelihood ratio test) and Blomberg’s K (with P based on randomisation test of the null hypothesis of K = 0).

Sperm trait PGLS Pagel’s λ Blomberg’s K

Estimate ( ± SE) t P λλPKP

Head length −0.05 ± 0.31 −0.172 0.864 1 < 0.001,1.00 1.0044 < 0.0001 0.8926 < 0.001 Midpiece length −0.05 ± 0.09 −0.505 0.614 1 < 0.001,1.00 1.0155 < 0.0001 1.6142* < 0.001 Flagellum length −0.06 ± 0.05 −1.118 0.265 1 < 0.001,1.00 1.0132 < 0.0001 1.5085 < 0.001 Total sperm length −0.05 ± 0.05 −1.089 0.277 1 < 0.001, 1.00 1.0129 < 0.0001 1.5175* < 0.001

* K also signiﬁcantly higher than 1 (P < 0.05).

5 T.C. Omotoriogun, et al. Zoology 140 (2020) 125770

Fig. 5. Variation in sperm total lengths and components in the Fringillidae, Muscicapidae, Turdidae and Hirundinidae with species represented in both Afrotropical and Northern temperate regions with barplot diﬀerentiated by colours (Afrotropical = red; Northern temperate = black) for the head, midpiece and ﬂagellum for the two regions. The phylogeny illustrates variation of sperm total length for the families with a scale bar of colour ranging from red to blue; the scale bar also indicates branch lengths in million years (myr).

Funding Declaration of Competing Interest

The study was supported by the Research Council of Norway (pro- The authors declare that they have no competing interests. ject no. 196554/V40 to JTL), The Quota Scheme in Lånekassen (ref. no. 2630465 to TCO) and International Foundation for Science (grant no. J/5724-1 to TCO), and grants from the Czech Science Foundation Acknowledgements (projects no. 17-24782S and 19-22538S to TA). MR was supported by the Research Council of Norway (project no. 230434). Funding orga- We thank those who assisted with field work during sperm collec- nizations had no role in the design of the study and collection, analysis, tion in Nigeria, Cameroon and other regions of the world. Terje and interpretation of data, or in writing the manuscript. Laskemoen and Gunnhild Marthinsen assisted with laboratory work. We appreciate the hospitality of Deni Bown during field work in IITA. We thank the A.P. Leventis Ornithological Research Institute for pro- viding field vehicle. This is contribution no. 154 from the A. P. Leventis Ornithological Research Institute.

Table 2 Phylogenetic least squares (PGLS) analysis of regional diﬀerences in sperm components of songbird species (N = 67) from four families with representation from both the Afrotropical and the Northern temperate region. The model including the maximum-likelihood of lambda (λ) value was compared against the models assuming λ = 1 and λ = 0, and superscripts following the λ values indicate probability (P) of likelihood-ratio of sperm component (ﬁrst position: against λ =0; second position: against λ = 1). The table also gives two estimate of the phylogenetic signal in the sperm components as expressed by Pagel’s λ (with P based on a likelihood ratio test) and Blomberg’s K (with P based on a randomisation test).

Family PGLS Pagel’s Lambda Blomberg’sK

Sperm trait Estimate ( ± SE) t P λλPKP

Fringillidae (N = 20) Head length 0.09 ± 0.23 0.438 0.666 0.996 < 0.0001, 0878 1.0216 < 0.0001 1.1941 0.006 Midpiece length −0.08 ± 0.67 −0.122 0.904 1 < 0.0001, 1 1.0307 < 0.0001 0.9448 0.003 Flagellum length −0.09 ± 0.29 −0.307 0.762 1 0.0005,1.00 1.0287 0.0005 0.9066 0.002 Total sperm length −0.07 ± 0.27 −0.261 0.797 1 0.0005,1.00 1.0284 0.0005 0.9218 0.001 Muscicapidae (N = 24) Head length −0.05 ± 0.08 −0.621 0.541 1 0.0005, 1 1.0952 < 0.0001 1.4670 0.001 Midpiece length −0.19 ± 0.17 −1.093 0.284 1 0.015, 1 1.0969 0.0001 1.1146 0.001 Flagellum length −0.13 ± 0.13 −1.002 0.327 1 0.0054, 1 1.0972 0.0002 1.0860 0.001 Total sperm length −0.12 ± 0.12 −1.044 0.308 1 0.0038, 1 1.0971 0.0002 1.0848 0.001 Turdidae (N = 13) Head length 0.15 ± 0.05 3.043 0.011 0 1, 0.0085 0.7044 0.0748 0.9254 0.052 Midpiece length 0.04 ± 0.14 0.304 0.767 1 0.035, 1 1.1699 0.0010 1.9568 0.003 Flagellum length −0.01 ± 0.08 −0.121 0.906 1 0.010, 1 1.1688 0.0006 2.0087 0.005 Total sperm length 0.00 ± 0.07 0.011 0.992 1 0.010, 1 1.1386 0.0008 2.0747 0.006 Hirundinidae (N = 10) Head length −0.01 ± 0.05 −0.280 0.787 1 0.059, 1 1.0680 0.0118 1.1467 0.004 Midpiece length −0.31 ± 0.26 −1.195 0.266 1 0.013, 1 1.0781 0.0007 1.1867 0.001 Flagellum length −0.22 ± 0.21 −1.051 0.324 1 0.026, 1 1.0784 0.0006 1.1543 0.002 Total sperm length −0.19 ± 0.19 −1.015 0.340 1 0.029, 1 1.0785 0.0005 1.1568 0.001

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